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Remission of the polycystic ovarian condition (PCO) in the rat following hemiovariectomy.

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THE ANATOMICAL RECORD 226:328-336 (1990)
Remission of the Polycystic Ovarian Condition
(PCO) in the Rat Following Hemiovariectomy
MARGARET CONVERY, GERALD F. McCARTHY, AND JAMES R. BRAWER
Department of Anatomy and The McGill Centre for the Study of Reproduction,
McGill University, Montreal, Canada
ABSTRACT
The estradiol valerate-induced polycystic ovarian condition in the
rat represents a normal ovarian response to aberrant endocrine stimuli. Although
we have shown that removal of one polycystic ovary (hemiovariectomy) results in
restoration of cyclicity and normal morphology in the remaining ovary by 1 week,
nothing is known about the process of recovery or about the role of the hypothalamopituitary unit in initiating recovery. We have therefore examined ovaries at 3, 12,
24,48, and 320 hours following removal of the contralateral polycystic ovaries. The
ovarian content and size distribution of healthy and atretic follicles was determined, a s well a s the occurrence of follicular cysts, type I11 large follicular structures, and corpora lutea. The plasma LH pattern was also examined a t a short
postoperative interval. At 3 hours, there was a significant increase in mean ovarian weight t h a t coincided with the emergence of healthy large secondary follicles.
By 12 hours, there was a significant sustained diminution in the number of atretic
follicles of all sizes, but the total number of healthy follicles did not increase
significantly until 120 hours. The cystic follicles had all but disappeared by 120
hours because of mechanical compression by newly developing ovarian tissue.
Ovarian recovery is, therefore, biphasic, consisting of a very early diminution in
atresia coincident with, and perhaps caused by, a major alteration in the plasma
LH pattern. The second phase is characterized by a wave of follicular recruitment
and development.
Anovulatory acyclicity characterized by ovaries containing multiple cystic follicles is a reproductive anomaly that occurs in a wide range of mammalian species
under a variety of circumstances. It is a major form of
infertility in the human, and it plagues the pork, cattle, and sheep industries. The polycystic ovarian condition (PCO) can be produced in the laboratory rat by a
variety of experimental manipulations, including constant light exposure (Daane and Parlow, 19711, anterior hypothalamic deafferentation (Halasz, 1969; Blake
et al., 1973), and neonatal androgen treatment (Gorski,
1971). We have generated a chronic PCO condition in
the rat by giving a single large dose of estradiol valerate (EV) (Brawer et al., 1978; Hemmings e t al., 1983;
Schulster e t al., 19841, and we are currently using this
model to explore the pathogenesis and the biology of
the polycystic ovary.
The ovaries in the EV-treated rat contain no corpora
lutea and few healthy secondary follicles. Large follicular cysts occur, which are characterized by a highly
attenuated membrana granulosa and a hypertrophied
thecal cell layer comprised of large polygonal, lipidfilled cells. Clusters of large polygonal secondary interstitial cells are abundant in the stroma (Hemmings et
al., 1983; Schulster et al., 1984; Brawer et al., 1986,
1989).
It appears that the expression of this characteristic
polycystic morphology does not reflect intrinsic ovarian pathology, but rather is the response of a n essentially normal ovary to a n abnormal pattern of gonado0 1990 WILEY-LISS, INC
tropin stimulation (Brawer et al., 1986; McCarthy et
al., 1986; Grosser e t al., 1987; Carriere e t al., 1989).
Examination of the histological transition from the
normal to the polycystic state (Brawer et al., 1986,
1989) indicates that the polycystic morphology is really
the product of complete or arrested atresia of developing secondary follicles and the absence of ovulation
(Brawer et al., 1986). The loss of a n ovulatory LH surge
accounts for the lack of corpora lutea, and the atresia of
secondary follicles results in the numerous clusters of
hypertrophied lipid-containing secondary interstitial
cells. The cysts themselves represent partial or arrested atresia of large secondary follicles or type I11
large follicular structures (Brawer et al., 1986, 1989;
Desjardins and Brawer, 1989). The processes of follicular recruitment and atresia that produce the polycystic ovary seems to be driven by a specific plasma gonadotropin pattern (McCarthy et al., 1986, 1987; Grosser
et al., 1987).
We have shown that cyclicity and normal ovarian
morphology can be restored in EV-induced PCO by the
removal of one polycystic ovary (i.e., hemiovariectomy)
(Farookhi et al., 1985), indicating that the polycystic
ovary retains the capacity for normal function. Regular
Received April 6, 1989; accepted June 20, 1989.
Address reprint requests to James R. Brawer, Department of Anatomy, 3640 University Street, Montreal P.Q. H3A 2B2, Canada.
REMISSION OF PCO
329
vaginal cyclicity and normal ovarian histology are re- Montreal, Quebec, Canada) and then housed one per
stored by 1 week following hemiovariectomy. The pres- cage. Atrial catheters were flushed with 50 IU of hepence of corpora lutea at this time point denotes the arin sodium (Heparin Sodium Injection U.S.P., Allen &
restoration of a gonadotropin surge. Interestingly, this Hanburys, Montreal, Quebec, Canada) diluted in 0.5
recovery process occurs in the absence of any signifi- ml of bacteriostatic (1.5%benzyl alcohol) 0.9%saline
cant change in the mean plasma gonadotropin concen- (Bacteriostatic Sodium Chloride Injection U.S.P.,
trations (Farookhi e t al., 1985). We have hypothesized, Squibb Canada) every 2-3 days thereafter.
After a postoperative interval of 1 week, three anitherefore, that hemiovariectomy interrupts the PCOassociated plasma LH pattern (McCarthy et al., 1986, mals were each placed a t 0400 into a n isolation cage. A
1987; Grosser et al., 19871, resulting in a new pattern 64 cm length of PE-100 tubing (Clay Adams, Parsippany, NJ; 0.034 inches in I.D. and 0.060 inches in O.D.)
that supports complete follicular development.
At present, however, we have no information on the was fed into the cage through the bore of a tightly
process of ovarian recovery from the polycystic state. coiled spring and attached to the catheter. The spring
Since we know that it is essentially complete by 1 week was held in place by a pivot a t the top of the cage that
following hemiovariectomy (Farookhi e t al., 19851, it is afforded the animal free movement during the samclear that during this short interval major changes in pling procedure. At this time, 1hour prior to sampling,
follicular dynamics occur resulting in development of the tubing and catheter (sampling line) were flushed
follicles to the preovulatory state as well as in disap- with 50 IU of heparin sodium diluted in 1 ml of bactepearance of the cysts. To determine how and why this riostatic 0.9%saline.
Beginning a t 0500 hours, 0.5 ml blood samples were
occurs, we have examined the changes in follicle populations at early intervals following hemiovariectomy drawn every 10 minutes over a 4 hour period. Each
in animals with PCO. In view of the significance of the blood sample was centrifuged for 2 minutes in a Beckplasma LH patterns in PCO (McCarthy et al., 1986, man Microfuge (15,600g). Two 100 p,1 plasma aliquots
1987; Grosser et al., 1987), we would predict that any were removed and frozen on dry ice. The remaining
change in follicular populations should be preceded by blood cells were resuspended in 200 p1 of bacteriostatic
a change in the plasma LH pattern. We have, there- 0.9% saline containing 10 IU heparidml. The resusfore, also examined the plasma LH pattern several pended blood cells were injected into the animal
hours after hemiovariectomy. Since we have found no through the sampling line a t the end of the next sam*correlation between the FSH pattern and the develop- pling interval. The samples from each animal were
ment and expression of the polycystic morphology (Mc- stored at -80°C for later determination of LH.
Carthy et al., 1986, 1987), we have not included obserSurgical Procedures
vations on the FSH pattern.
At 0900 hours the animals were removed from the
MATERIALS AND METHODS
isolation cages, anesthetized with ether, and the right
Plasma LH Patterns
ovary of each animal was removed through a flank
incision. At 1200 hours the animals were returned to
Animals and treatment
the sampling cages and were prepared for serial blood
Twelve young, female Wistar rats (145-200 g) were sampling. At exactly 1300 hours (4 hours posthemiobtained from Charles River Ltd. (St. Constant, Que- ovariectomy) 0.5 ml blood samples were withdrawn at
bec, Canada). They were housed in groups of four and 10 minutes intervals over a 4 hour period a s described
were given free access to pelleted rat food and water. above.
The animals were exposed to 14 hours of light daily
The remaining (unsampled) three animals were
(lights on a t 0700 hours), and their estrous cycles were sham operated a t 0900 hours to serve as controls for the
monitored by daily examination of vaginal smears. hemiovariectomized group. At 1000 hours each animal
Only those animals that displayed two consecutive nor- was placed into a n isolation cage and prepared for semal 4 day estrous cycles prior to treatment were used rial blood sampling. One hour later (2 hours after sham
in the study.
operation) the animals were sampled as described
Each animal was injected (i.m.) with a 2 mg dose of above. The samples from each animal were stored a t
estradiol valerate (EV) (Delestrogen U.S.P., Squibb -80°C for later LH determinations.
Canada Inc., Montreal, Quebec, Canada) dissolved in
0.2 ml sesame oil. Three to 4 weeks thereafter, all an- LH assay
imals displayed persistent vaginal cornification. Only
The concentrations of LH were determined by radiothose animals that continued to show unequivocal and immunoassay using 100 pl samples. Samples (pre- and
sustained vaginal cornification by week 7 after injec- posthemiovariectomy and of the sham-operated anition were selected for the study.
mals) were assayed in singleton for LH in separate
assays. Assay kits were obtained from the National
Catheter placement and serial blood sampling
Pituitary Agency (NIAMDD, Bethesda, MD). The
The six animals that had been selected a t week 7 NIAMDD reference preparation used for the assays
were each fitted with a chronic indwelling atrial cath- was LH-RP-2. The assay procedures have been deeter using a modification (Grosser et al., 1987) of a scribed previously (Grosser et al., 1987). The intraastechnique previously described by Tannenbaum and say coefficient of variation in the LH assay for the preMartin (1976). Following the surgery, each animal was and posthemiovariectomy samples a t the 20%, 50%,
given a n intramuscular injection of 0.35 ml of 300,000 and 80%level of counts for a n arbitrary dose relative to
IU/ml penicillin G procaine (Ayercillin, Sterile Penicil- that of a zero dose (BIBo) was 4.9%,3.1%, and 10.4%,
lin G Procaine Suspension U.S.P., Ayerst Laboratories, respectively. The limit of detection for LH was 105 pg/
330
M. CONVERY ET AL.
ml. The intraassay coefficient of variation for the LH
assay used for samples from sham-operated animals
was 8.8%, 6.3%, and 26.3%, respectively, at the 20%,
50%, and 80% level of counts (B/Bo). The limit of detection of LH in this assay was 70 pg/ml.
Data analysis
LH pulses were defined, for the most part, using the
criteria established by Gallo (1981) and later modified
by Ellis and Desjardins (1982). In our pulse analysis,
a n LH determination and associated coefficient of variation (CV) was assigned only to the lowest-most point
(nadir) of the ascending phase of a potential pulse. An
LH value was also assigned to the highest-most point
(peak) of a potential pulse. A pulse was defined when
the peak value was greater than the nadir value plus
two times the CV of the nadir value. The lowest-most
point of the descending limb of a pulse, as long as it
differed from the peak value plus twice its associated
CV, marked the end of a pulse. Pulse nadir, peaks,
amplitudes, and frequencies were each determined for
the 4 hour sampling period and were expressed as the
mean ? the standard error of the mean.
Morphology
Treatment of animals
Young female Wistar rate, weighing between 145
and 200 g, were purchased from Charles River Ltd. The
animals were maintained under conditions of lighting
described above. Estrous cycles were monitored for 2
weeks by daily examination of vaginal smears. Animals each exhibiting at least two normal 4 day cycles
were used in this study. Each animal was lightly anesthetized with ether and given a n intramuscular injection of 2.0 mg EV dissolved in 0.2 ml sesame oil.
Eight weeks after the EV treatment, a group of 30
animals each demonstrating unequivocal and sustained cornified vaginal smears during the 2 prior
weeks were chosen for the study. Animals were anesthetized with ether, and the right ovary from each animal was removed through a flank incision. The ovaries were examined to confirm that the animals used in
the study did, in fact, have PCO a t the time of ovariectomy. Several such ovaries contained corpora lutea and
hence disqualified the animals from the study, leaving
only four (out of the six) animals in one of the groups.
In order that the sample number be consistent, we have
used only four animals, with confirmed PCO, from each
postoperative time point.
Groups of four animals were killed by decapitation at
3,12,24,48, and 120 hours after hemiovariectomy. The
remaining ovary was removed, weighed, and fixed in
Bouin’s solution for 24 hours. The oviducts were removed and examined for the presence of ova. Following
fixation, the ovaries were dehydrated and embedded in
paraffin. Each ovary was serially sectioned a t 7 pm,
and the sections were stained with hematoxylin and
eosin (Brawer et al., 1986). Four polycystic ovaries removed a t the time of hemiovariectomy were prepared
as described above.
Analysis of follicle populations
The procedure for the analysis of follicle populations
used in this study is based on a previously published
methodology (Brawer et al., 1986). All primary and
secondary follicles in each ovary were measured using
a n ocular micrometer. Measurements were made in the
section of the follicle that contained the nucleus and
nucleolus of the ovum. For each follicle, two perpendicular diameters were measured. Each diameter was
measured from basement membrane to basement
membrane, and the average of these two measurements was then calculated. In addition to size designation, each follicle was classified as either healthy or
atretic. A follicle was considered atretic if it exhibited
degeneration of the ovum or one or more pyknotic granulosa cells.
Follicle size distribution histograms were generated
for each postoperative time point. The follicles at each
time point were grouped into three size categories for
the purpose of statistical analysis. These are follicles
with diameters less than 250 pm (mostly primary and
very early secondary follicles), follicles with diameters
between 250 and 500 pm (secondary follicles), and follicles with diameters greater than 500 pm (large secondary follicles, including putative preovulatory follicles). Multigroup comparisons of the data were
analyzed by one way analysis of variance. Comparison
of means between two groups was evaluated by using
Student’s t test. Differences were considered significant at P < 0.05.
The larger follicular structures such as cystic follicles were identified and counted a t each posthemiovariectomy time point. Cystic follicles were identified according to established criteria (Brawer et al., 1986,
1989; Desjardins and Brawer, 1989).
Type I11 large follicular structures (Brawer et al.,
1989; Desjardins and Brawer, 1989)were distinguished
from large secondary follicles by virtue of the fact that
they did not contain ova, although occasionally a very
degenerate circular structure, possibly a deteriorated
ovum, occurred floating free in the large antrum. The
membrana granulosa was thick and often plicated.
Also, in contrast to large secondary follicles, large polygonal cells were frequently seen interspersed among
the smaller fusiform variety in the theca interna.
RESULTS
Plasma LH Patterns
The LH patterns for the nonoperated controls are
shown in Figure 1A-C. The patterns in these animals
are identical to the PCO-associated LH pattern described previously (Grosser et al., 1987). The mean nadir, peak, and amplitude of LH pulses in the nonoperated controls were 123 S 18, 204 ? 35, and 78 2 45
pg/ml, respectively. The frequency was approximately
one pulsehour. The plasma LH patterns displayed by
the three sham-operated controls (not illustrated) indicate that the procedure itself may cause a significant
suppression of LH pulsatility. Two of the three shamoperated controls showed no detectable LH pulses over
the sampling interval, while the third animal exhibited three pulses of amplitude and duration comparable
to those characterizing pulses in nonoperated controls.
Within the 4 to 8 hour interval following hemiovariectomy, the plasma patterns of LH differed markedly
from those observed prior to hemiovariectomy (Fig.
1D-F). The mean nadir, peak, and amplitude of pulses
in the hemiovariectized animals were 248 L 93,406 2
331
REMISSION O F PCO
6ool
400
A
6oo]
B
6o01 D
,:I
OJ,
6ool
16000
.
*
,
,
1
I
80001
01 I
0
,
i
. , . . . . : . . j
O
,
F
1
400
2o-l
,
0
24000
r
I
80
160
240
0
80
160
240
Fig. 1. Plasma LH patterns. A-C: LH patterns prior to hemiovariectomy in three individual animals with PCO. D-F: LH patterns in
the same animals 4 hours after hemiovariectomy. The scale in F differs from the others to accommodate a very large LH episode. As a
result, the smaller pulses cannot be resolved in this panel. All apparent pulses meeting the criteria described in Materials and Methods
are indicated by a n asterisk.
150, and 194 k 130 pg/ml, respectively. The pulse frequency was similar to that observed in the nonoperated
control patterns (about one pulsehour), as were the
durations and shapes of the pulses.
3.68
0.45, 3.33
0.54, and 5.02
0.48 g, respectively. In each group, the ovaries removed after hemiovariectomy were significantly heavier than their respective controls (removed at hemiovariectomy) (P <
0.05).
Cyclicity
All animals at the time of hemiovariectomy exhibited persistent vaginal cornification. This condition
was also observed in all animals at the 3,12,24, and 48
hour postoperative intervals. By 120 hours, there was
considerable variation in the smear patterns. The
smears of two animals exhibited all cell types, one exhibited a n estrous-diestrous smear, and one exhibited a
proestrous-estrous smear. No ova were observed in the
oviducts at any postoperative time point.
Ovarian Weights
The weights of the ovaries removed at the various
time points were compared with those removed at hemiovariectomy (controls). The mean weight of control
(polycystic) ovaries ( C SEMI removed at hemiovariec0.370 g. The mean ovarian weight
tomy was 1.57
3 hours following hemiovariectomy (2.505 0.540 g)
was significantly greater than the control weight
(P < 0.05). The mean ovarian weights a t 12,24,48, and
120 hours posthemiovariectomy were 3.37 k 0.70,
*
*
*
*
Ovarian Content of Corpora Lutea, Cysts, and Type 111
Large Follicular Structures
Corpora lutea were absent from control ovaries and
from ovaries removed at 3 and 12 hours following hemiovariectomy. At 24 hours, one or two corpora lutea
appeared in three (out of four) ovaries, and a t 48 hours
two of the ovaries showed one or two corpora lutea.
Three of the ovaries removed at 120 hours each exhibited four to six large corpora lutea, whereas one ovary
contained only one corpus luteum. Smaller clusters of
luteinized tissue, possibly resulting from luteinization
of small secondary follicles also occurred in each ovary
(see Fig. 6).
Type I11 large follicular structures (Figs. 2,3A) were
easily identified on the basis of previously established
criteria (Brawer et al., 1989; Desjardins and Brawer,
1989). The use of serial sections in the present study
allowed us to confirm our suspicion that type I11 large
follicular structures do not contain ova. An average of
332
M. CONVERY ET AL.
Fig. 2. Type 111 large follicular structure. The large antrum is surrounded by a thick membrana granulosa, which is plicated along the
right side of the field. x 100.
six type I11 large follicular structures occurred in each
control ovary removed a t hemiovariectomy. This number declined to a n average of two per ovary a t 12, 24,
and 48 hours. By 120 hours posthemiovariectomy, only
one of four ovaries showed a single type I11 large follicular structure.
Seven to 10 cystic follicles (Figs. 3B, 4) occurred in
each ovary removed a t 3, 12, and 24 hours after hemiovariectomy. At the 48 hour interval, each ovary contained between four and seven cysts, and a t 120 hours
cysts were absent from all but one ovary, which contained only two cystic follicles. The cysts a t the later
time points appeared squeezed and distorted by developing ovarian tissue (Fig. 5). This process of compression was progressive in time, ultimately resulting in
the collapse and disappearance of most cysts by 120
hours (Fig. 6).
Ovarian Follicle Content
The mean number of healthy and atretic follicles
(Fig. 3C) in control (polycystic) ovaries (time 0) and in
ovaries removed at 3 hours posthemiovariectomy were
similar (Fin. 7). At 12 hours there was a reduction in
the
number Of follicles largely because Of a decrease in the population of atretic
There was
little change a t 24 and 48 hours, but at 120 hours the
Fig. 3.The wall of A: a type 111 large follicular structure, B: a cyst,
and C: an atretic secondary follicle. In each case, the antral surface of
the membrana granulosa is indicated by arrowheads. x 400.
Fig. 4. Polycystic ovary charaterized by numerous cystic follicles
and by a n absence of corpora lutea and of healthy secondary follicles.
The hilus appears in the upper right of the field. X 17.
Fig. 6. Ovary removed 120 hours after hemiovariectomy exhibiting
the full complement of follicles that typifies the normal ovary. Several
corpora lutea are also evident. x 17.
0
Fig. 5. Ovary removed 48 hours after removal of contralateral ovary
(hemiovariectomy).Two residual cysts (stars) are compressed and distorted by developing follicles and only one cyst (triangle) maintains
the characteristic appearance. The hilus appears in the right of the
field. ~ 1 7 .
3
12
24
TIME (hrs)
48
120
Fig. 7. Total ovarian content of follicles a t the different posthemiovariectomy intervals. The mean ( 5 SEMI number of healthy follicles
(of all sizes) a t each posthemiovariectomy time point is depicted by the
stippled bars. The mean ( 2 SEM) number of atretic follicles are represented by the striated bars at the top,
Size Distribution of Follicles
mean number of follicles exceeded that seen in controls. The mean number of atretic follicles/ovary remained relatively constant from 12 to 120 hours, but
there was a marked increase in the population of
healthy follicles.
The mean follicular content of the ovaries included
follicles in all phases of development from the early
primary (small) to the late secondary (large) stage. The
data have been further broken down to analyze the
follicular size distribution at each postoperative inter-
334
M. CONVERY ET AL.
"1
40
A
48 hrs
0 hrs
1-/
40
1
20
0
U
LL
LI
60
B
W
1 2 hrs
80
D
1 2 0 hrs
m
52
60
40
40
20
20
0
,
0
.
100
50
'
150
200
.
250
.
300
.
350
.
400
.
.
500
450
0
200
100
)
50
150
300
250
500
400
350
450
FOLLICLE DIAMETER (,urn)
@
Fig. 8. Size distribution of healthy and atretic follicles at four posthemiovariectomy time points. The
mean ( 2 SEMI number of follicles in the size ranges indicated on the X axes are depicted. Healthy
follicles are indicated by the stippled bars. and the atretic follicles are designated by the cross-hatched
bars a t the top.
val. This permitted the evaluation of the contribution
of the different follicular populations to the total mean
number at each time point.
Size distributions of follicles with diameters less
than 500 pm are presented in Figure 8. Figure 8A
shows the size distribution of follicles in the polycystic
(control) ovaries. The pattern at 3 hours posthemiovariectomy (data not shown) was essentially identical. At
12 hours (Fig. 8B), however, there were significant deviations from the control pattern. There was a significant decrease in the number of atretic follicles of all
sizes (P < .001), and this low level of atresia was maintained throughout the duration of the study. Although
there were fewer primary follicles (diameter <250 pm)
at 12 hours than in controls, the difference was not
significant. However, a significant increase in this population occurred between 12 (Fig. 8B) and 48 (Fig. 8C)
hours, and this trend continued such that the number
of these small follicles (diameter <250 pm) was significantly greater at 120 hours (Fig. 8D) than in controls.
No significant changes were observed in follicles with
diameters of 250-500 (small-medium secondary follicles) at any of the postoperative time points.
The control ovaries contained no healthy large sec-
181
0
3
12
24
TIME (hrs)
48
120
Fig. 9. Ovarian content of large secondary follicles a t the different
posthemiovariectomy intervals. The mean ( 5 SEM) numbers of
healthy large secondary follicles (diameter > 500 km) a t each time
point is depicted by the stippled bars. The mean (? SEM) numbers of
atretic large secondary follicles are designated by the striated bars.
REMISSION OF PCO
ondary follicles (Fig. 9). By 3 hours, however, each
ovary contained a n average of six such structures. Another significant increase in this population occurred
between 3 and 24 hours (P < 0.001), after which no
further change occurred. There were no significant differences in the number of atretic large secondary follicles between any of the time points.
335
sizes between 0 and 12 hours is a key event in the
process of recovery. Atresia plays a major role in the
development of the polycystic condition (Brawer et al.,
1986). The process of atresia is responsible for the low
overall content of follicles in the polycystic ovary, as
well a s the dearth of healthy large secondary follicles.
Moreover, it appears that the cysts themselves are
products of arrested atresia (Brawer et al., 1986). It is,
DISCUSSION
therefore, not surprising that the recovery from the
Within several hours following hemiovariectomy, polycystic condition is preceded by a reversal of the
marked changes occur in both the plasma LH pattern trend favoring atresia to one permitting the normal
and in the histology of the polycystic ovary. The LH full range of follicular development.
patterns were determined from 4 to 8 hours following
Although the polycystic morphology appears to rethe procedure, leaving open the question as to whether sult from a predominance of atresia, the attenuation of
alterations in the LH pattern may actually occur a t a n atretogenesis is insufficient, by itself, to account for
even earlier time point. This is, however, a likely pos- full ovarian recovery. There is a significant hiatus besibility, since significant changes in ovarian weight tween the reduction of atresia (at 12 hours) and the
and histology are apparent a s early a s 3 hours posthe- increase in the total number of healthy follicles obmiovariectomy. Hemiovariectomy produced marked served at 120 hours. Indeed, the ovarian content of
increases in nadir, peak, and amplitude of LH pulses healthy follicles a t 12 hours is less than in polycystic
despite the fact that anesthesia andlor surgical stress controls because of reduction in the number of healthy
alone resulted in a suppression of LH pulses. Clearly, primary and small secondary follicles a t 12 hours. The
remove1 of one polycystic ovary caused abrupt and pro- relatively stable ovarian follicular content observed benounced feedback responses at the hypothalamopitu- tween 12 and 48 hours can be attributed to a lag beitary level. Whether this rapidly induced modulation of tween the diminution in atresia and a n increment in
gonadotropin release is the principle causal antecedent follicular recruitment. By 120 hours both of these pheto the process of ovarian recovery remains to be deter- nomena contribute to the enhanced ovarian content of
mined. It does, however, seem probable in view of pre- follicles.
vious evidence linking a specific LH (but not FSH) patThus at least two processes contribute to the recovtern to the onset and maintenance of PCO in the EV ery of the polycystic ovary. The LH pattern is probably
treated rat (McCarthy et al., 1986, 1987). Moreover, the principle determinant for the level of atresia. A
alteration of the PCO-associated LH pattern by means second event, however, must account for the accelerof treatment with the opiatergic antagonist naltrexone ated folliculogenesis. This may, in fact, be a further
results in significant recovery of the polycystic ovaries modification of the LH pattern, or it may involve a late
within 3 days (Carrier et al., 1989).
posthemiovariectomy change in the FSH pattern. AlCoincident with the rapid change in the plasma LH ternatively, the stimulus may be intraovarian, derivpattern are significant changes in the ovaries. Ovaries ing from the new population of large healthy secondary
removed at the 3 hour posthemiovariectomy time point follicles. The nature of the folliculogenic stimulus reweighed significantly more than the polycystic con- mains to be determined.
trols. Moreover, they contained a n abundance of
The disappearance of cystic follicles appeared to be
healthy large secondary follicles, which were totally caused by progressive mechanical compression resultabsent in control ovaries. Since neither the content nor ing from the development of, first, large secondary folthe size distribution of the other follicular structures licles and later, follicles of all sizes. Follicular cysts,
changed from the 0-3 hour time period, the increase in therefore, do not appear to respond actively, a s do other
ovarian weight is most probably due to the emergence populations of follicles or follicular structures, to hemiof the healthy large secondary follicles.
ovariectomy, but rather are eliminated by passive conIf, as suggested, alterations in the plasma LH pat- striction. This is consistent with our earlier observatern contribute significantly to the changes in ovarian tions that cyst development coincides with a loss of
histology, the rather sudden appearance of healthy receptivity to LH (Brawer et al., 1989), suggesting that
large secondary follicles may be causally linked to the the cystic follicle is a relatively inert structure.
rapid onset of the relatively large amplitude LH pulses
One of the most intriguing follicular structures
following hemiovariectomy. We have shown that the unique to the polycystic ovary is the type I11 large foldevelopment of the polycystic ovarian morphology is licular structure. These are comprised of a membrana
preceded by a loss of high amplitude LH pulses occur- granulosa and thecal cell layers similar to those charring between 16 and 21 days following EV treatment acterizing Graafian follicles. The type I11 large follicu(McCarthy e t al., 1989). The resultant plasma LH pat- lar structure, however, differs from a Graafian follicle
tern, which is maintained from 21 days onward, is that in that it is often larger, exhibits mitotic figures in the
typifying the established PCO condition (Grosser et. perimural granulosa cells (Brawer et al., 1989; Desjaral., 1987). Interestingly, the incidence of atresia, partic- dins and Brawer, 1989), and, a s demonstrated by the
ularly among large secondary follicles, is maximal at present study, contains no ovum. Paradoxically, i t is
this post-treatment interval (Brawer et al., 1986). One the only follicular structure in the polycystic ovary in
might predict, therefore, that the reintroduction of which granulosa cells bind LH (hCG) (Brawer et al.,
large amplitude LH pulses would rescue a cohort of 1989), and it is, therefore, the only structure that could
developing follicles from atresia.
be readily luteinized following a n LH surge. Two pieces
The marked reduction in atresia in follicles of all of evidence suggest that the corpora lutea, which begin
336
M. CONVERY ET AL.
to appear a t 24 hours following ovariectomy, derive
from these structures. First, the incidence of corpora
lutea increases as that of type I11 large follicular structures declines. Second, ova were never found in the
oviducts, indicating that the structures giving rise to
the corpora lutea did not, or could not, ovulate. The
nature, function, and role of the type I11 large follicular
structure in PCO remain to be elucidated.
In summary, the poverty of follicles in general, and
of healthy large secondary follicles in particular, in the
polycystic ovary appears to be the result of a constant
rate of follicular recruitment together with a high level
of atresia. Hemiovariectomy reverses both of these
trends and produces a rapid decline in atresia, followed somewhat later by enhanced follicluar recruitment. The resultant newly developing ovarian tissue
compresses and eventually destroys the cysts. The diminution in atresia that initiates the recovery process is
coincident with the restoration of high amplitude LH
pulses.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the technical assistance of Ms. Dalia Chen. This work was supported
by a n operating grant to J.R.B. from the Medical Research Council of Canada.
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